Proteoglycan (PG) aggrecan is a major macromolecular component of articular cartilage. PG has been considered a T-cell autoantigen in patients with rheumatoid arthritis (RA), an autoimmune disease of the peripheral joints that results in inflammatory destruction of cartilage and loss of joint function. The arthritogenic potential of PG is underscored by the observation that immunization of BALB/c mice with human PG induces arthritis with clinical and immunological features similar to those in RA. We have recently found that PG extracted from adult human cartilage is citrullinated. While citrullination (enzymatic conversion of arginine residues to citrulline) is a post-translational protein modification that may occur in healthy individuals, the majority of patients with RA, unlike healthy subjects, recognize citrullinated self proteins as foreign and produce anti-citrullinated protein autoantibodies (ACPA). Importantly, we have found that ACPA+ RA sera react with in vivo or in vitro citrullinated human PG, predominantly with the first globular (G1) domain of the molecule. Citrullinated PG present in the joint cartilage of RA patients has the potential to initite autoimmune responses and/or serve as a target for ACPA with serious consequences. However, very limited data is available about T-cell recognition of citrullinated PG epitopes, and nothing is known about the epitope repertoire, cartilage-specific deposition, and pathogenicity of PG-specific ACPA in RA. The studies described in Aim 1 will (i) explore the citrullinated PG (G1 domain)-specific T-cell and ACPA epitope repertoire using peripheral blood from RA patients, and (ii) identify PG-specific ACPA deposited in cartilage of damaged RA joints. Since mice with human PG-induced or PG G1-domain induced arthritis (GIA) also produce ACPA, this animal model provides a unique research tool for investigating the mechanistic aspects of citrullinated PG-specific autoimmunity that cannot be studied in patients with RA. Therefore, in Aims 2 and 3, we will (i) map the full repertoire of citrullinated epitopes within the G1 domain of human PG in BALB/c mice immunized with in vitro citrullinated G1, (ii) monitor the evolution of citrullinate G1-specific immunity, (iii) correlate the magnitude and epitope repertoire of anti-G1 ACPA with the severity of arthritis, and (iv) determine whether selective loss of citrullinated G1-specific immune tolerance can lead to arthritis development. The results of our studies should provide clinically relevant insights into the role of citrullinated PG in the pathogenesis of ACPA+ RA.